The long term goal of the proposed research is to understand in detail the biochemical mechanism of trans-splicing in the parasitic nematode, Ascaris. Cell free extracts prepared from Ascaris embryos efficiently catalyze cis and trans-splicing. Both processing reactions require the participation of several small nuclear RNAs (U snRNAs) and conditions have been established in which cis and/or trans-splicing activity in vitro can be made dependent upon the addition of any of five individual synthetic snRNAs. The studies proposed here will use this reconstitution system to determine functionally relevant sequences and structures of specific U snRNAs. Particular emphasis is given to U6 and U2 snRNAs because these RNAs are unambiguously present in catalytically active cis and trans- spliceosomes and because they may participate directly in the catalysis of splicing. Five specific aims are proposed: a) To identify functionally important backbone positions (including possible metal-coordination sites) in U6, phosphorothiate substitution interference will be employed. b) To obtain a complete picture of nucleotides required for U6 function, base-specific chemical modification interference analysis will be used. c) To identify functionally significant sequence elements in U2 snRNA, it will be subjected to scanning block-mutagenesis. d) To clarify the mechanism of splice-site recognition and juxtaposition in trans-splicing, site-specific crosslinking (via incorporation of thio uridine at or near splice sites) will be performed. e) To obtain a detailed understanding of snRNA-snRNA as well as snRNA-substrate interactions in cis and trans-splicing, a comprehensive crosslinking analysis using short wave length UV, psoralen and site or region specific substitution with thiouridine will be initiated. In combination, these approaches may provide significant new mechanistic insight into trans-splicing and as a consequence clarify its relationship to cis-splicing. Furthermore, transsplicing is a key step in mRNA maturation in a variety of medically important human parasites including nematodes, trypanosomes, and Schistosomes. A thorough understanding of this unusual RNA processing reaction may suggest novel strategies for therapeutic intervention in these parasitic diseases.